The goal of our research group is to elucidate the molecular mechanisms underlying the initiation phase of protein synthesis in eukaryotic organisms. We use the yeast saccharomyces cerevisiae as a model system and employ a range of approaches - from genetics to biochemistry to structural biology - in collaboration with Alan Hinnebusch and Tom Devers labs at NICHD and several other research groups around the world. Eukaryotic translation initiation is a key control point in the regulation of gene expression. It begins when an initiator methionyl tRNA (Met-tRNAi) is loaded onto the small (40S) ribosomal subunit. Met-tRNAi binds to the 40S subunit as a ternary complex (TC) with the GTP-bound form of the initiation factor eIF2. Three other factors eIF1, eIF1A and eIF3 also bind to the 40S subunit and promote the loading of the TC. The resulting 43S pre-initiation complex (PIC) is then loaded onto the 5-end of an mRNA with the aid of eIF3 and the eIF4 group of factors the RNA helicase eIF4A; the 5-7-methylguanosine cap-binding protein eIF4E; the scaffolding protein eIF4G; and the 40S subunit- and RNA-binding protein eIF4B. Both eIF4A and eIF4E bind to eIF4G and form the eIF4F complex. Once loaded onto the mRNA, the 43S PIC is thought to scan along the mRNA in search of an AUG start codon. This process is ATP-dependent and likely requires multiple RNA helicases, including the DEAD-box protein Ded1p. Recognition of the start site begins with base pairing between the anticodon of tRNAi and the AUG codon. This base pairing then triggers downstream events that commit the PIC to continuing initiation from that point on the mRNA. These events include ejection of eIF1 from its binding site on the 40S subunit, movement of the C-terminal tail (CTT) of eIF1A, and release of phosphate from eIF2, which converts it to its GDP-bound state. In addition, the initiator tRNA moves from a position that is not fully engaged in the ribosomal P site (termed P(OUT)) to one that is (P(IN)) and the PIC as a whole converts from an open conformation that is conducive for scanning to a closed one that is not. At this stage eIF2GDP dissociates from the PIC and eIF1A and a second GTPase factor, eIF5B, coordinate joining of the large ribosomal subunit to form the 80S initiation complex. eIF5B hydrolyzes GTP, which appears to result in a conformational reorganization of the complex, and then dissociates along with eIF1A. Advances in understanding the mechanism of start codon recognition In collaboration with the labs of Venki Ramakrishnan (MRC, UK) and Alan Hinnebusch (NICHD) we used cryo-electron microscopy to determine structures of the PIC in several different states before and after start codon recognition. These structures revealed important details of the architecture of the PIC and elucidated conformational changes that take place in response to recognition of the start codon in the mRNA. Movement of the tRNA from a state not fully engaged in the P site of the ribosome (Pout) to one fully engaged (Pin) can be seen clearly by comparing the structures before and after start codon recognition. This tRNA movement creates steric and electrostatic clashes with the key gatekeeper protein eIF1 that cause the factor to deform and shift position on the 40S ribosomal subunit, likely facilitating its release from the PIC. In addition, the beta subunit of eIF2 disengages from eIF1 upon start codon recognition, which probably further destabilizes eIF1s binding to the complex. In addition, we can now see domains of the large, heteromultimeric factor eIF3, which binds both to the back face of the 40S subunit, as expected, but also reaches around to the interface side to interact with several of the factors bound there. These structures explain the molecular basis for many of the results from biochemical and genetic studies done in our labs and by others, and will serve as a framework on which to base future experiments to increase our understanding of eukaryotic protein synthesis and its regulation.